Cancer remains the second leading cause of death in the world, mostly due the metastatic spread of tumor cells to distant sites. The molecular mechanisms and factors involved in metastasis have not been fully characterized, which leads to an inability to prevent metastasis. Metastasis is exacerbated not only by tumor- specific signals, but also systemic signals, including hormones and peptides associated with normal pregnancy. Identifying and understanding both the tumor-derived and systemic factors involved in tumor metastasis remains an intense area of research. One such factor is adrenomedullin (AM), which is a multifunctional peptide well-documented as being elevated during human pregnancy and numerous cancers. High AM during cancer is associated with distant metastasis and poor patient prognosis, but the specific mechanisms of AM on metastasis are not well understood. Published reports demonstrates that AM is essential for lymphatic development and has anti-inflammatory properties. We recently showed that high tumor-derived AM is correlated with tumor lymphangiogenesis and metastasis. Therefore, the proposed experiments in this grant will attempt to elucidate i) how tumor-derived AM induces lymphangiogenesis within the tumor microenvironment, ii) how systemic AM dosage during pregnancy alters tumor vascularization and metastasis, and iii) whether systemic AM suppresses immune surveillance. Specific aim 1 will determine if tumor-derived AM acts indirectly through macrophages to promote lymphangiogenesis. Macrophages will be isolated from AM dosed tumors using magnetic beads and assessed by flow cytometry, immunohistochemistry, and quantitative RT-PCR for changes in macrophage recruitment, reprogramming, and expression of lymphatic-specific growth factors, which would all contribute to the observed lymphangiogenesis. Specific aim 2 will test if systemic AM alters lymphangiogenesis and/or immune activation, both of which could influence metastasis. Labeled tumor cells will be implanted into mice with a genetic dosage of AM. These mice will be characterized for lymphangiogenesis and metastasis using the methods mentioned above. The use of virgin and pregnant mice from each genotype will identify if high systemic AM during pregnancy is important in the reported enhanced metastasis during pregnancy. To assess one component of immune activation, AM-treated natural killer cell's cytokine production and cytotoxicity will be characterized using ELISA and flow cytometry. The completion of these aims will help i) elucidate the local and systemic roles of AM on tumor metastasis, ii) implicate high AM levels during pregnancy as a contributing factor in pregnancy-associated metastases, and iii) further our understanding of AM's roles on innate immunity. Together, this research could further verify the AM signaling system as a pharmacologically-tractable target to reduce tumor metastasis.